The present invention relates generally to the field of bio-technologies and, more particularly, to microbiology and environmental protection, refers to the creation of a novel pool for cleaning oil-polluted soil, water and equipment, to the manufacture of a new biological preparation based on this pool designed to be used for the purification of water, industrial sewage, soil and other media from a wide range of petroleum products and directly from crude oil, and to the use of the obtained biological preparation for the efficient recovery of the above said oil-polluted media.
The ever-growing volumes of oil output, transportation, refining and utilization result in the wide-spread pollution of water and soil areas with crude oil and oil products. Despite safety precautions taken, accidents intermittently occur at petroleum-extracting and oil-refining enterprises, pipelines, oil tankers, resulting in an environmentally hazardous oil and oil-product spillage. It is common knowledge that Nature itself possesses an efficient system for the recovery of oil-polluted areas by means of microorganisms capable of assimilating the hydrocarbons of oil. However, the microflora of Nature is no longer capable of effectively recovering million tons of oil finding every year its way into the environment. As a result, the mankind has found itself under the real threat of ecological disaster (see, L. R. Brown. Chemical Engineering Progress. (1987) Vol. 83, No. 10, pp. 35-40; xe2x80x9cOil-degrading microorganismsxe2x80x9d).
Such traditional methods of recovery as mechanical, chemical and physical fail to provide a sufficient degree of recovery of oil-polluted water and soil. Besides, in many cases the application of these methods inflicts a more pronounced damage on the environment than oil spillage itself.
In view of the above, biological methods of recovery of oil-polluted media recently find the ever-growing application, including the use of oil-oxidizing microorganisms. Analysis of the development of bio-technologies relating to the recovery of oil-polluted water and soil ecosystems and equipment enables one to come to the conclusion that the processes based on the oil-product biodegradation under the action of microorganisms are ecologically efficient. In accordance with known bio-technologies, the polluted medium may be exposed to the action of biological preparations, including microorganisms either in the form of pure isolated cultures or pools of microorganisms, that is to say a combination or association of two or more organisms. However, it should be emphasized that from the viewpoint of achieving of a more complete utilization of biotechnological functionality of microorganismsxe2x80x94oil biodegrading agents, the application of pure isolated cultures of microorganisms is less advantageous than that of mixtures or associations of microorganisms which, in virtue of their specificity, are capable of utilizing a wider range of compositionally heterogeneous substrates (industrial sewage, soil pollution, etc.) and provide degradation of organic compounds at the expense of a combined action of several types of microorganisms. Thus a more pronounced ecological efficiency of biotechnological decontamination and recovery is attained, said biotechnological decontamination and recovery being carried out for a shorter time.
As a rule, known biological preparations for the recovery of oil-polluted areas are characterized either by a relatively low efficiency in virtue of the selectivity of monospecies cultures used in the biological preparation or by complexity of producing the biological preparation, which is associated with the process for adapting microorganisms to the co-existence when using a microorganism pool in the biological preparation concerned.
Thus, there has been known the strain Phodococcus sp. HX7 possessing oil-oxidizing properties, which is used when cleaning water and soil from oil pollution. In the treatment of the polluted areas with this bacteria, the usual dose of application is 1 gram of the cellular biomass per square meter of a treated area; or in the case of using a suspension of the biological preparation based on the above-cited bacteria it contains 1xc2x7107-1xc2x7107-1xc2x7108 cells in 1 milliliter (1xc2x7107-1xc2x7108 cells/ml). In so doing, the application rate of the suspension is 1 liter per square meter (1 l m2) of an oil-polluted area.
However, the above-mentioned process for cleaning media from oil-pollution is insufficiently effective, since the use is made of a bacterial monospecies which limits a range of hydrocarbons to be purified. Besides, a process for growing such a culture is of long duration and may take as long as several days (see, Russian Patent No. 2,039,714, IPC C 02F 3/34, 1995).
Known in the prior art is the natural strain Pseudomonas putida-36 and its utilization for the recovery of oil-polluted soil (see, U.S. Pat. No. 4,822,490, IPC C 02F 3/34, 1989). In the known method of cleaning water and soil, a bacterial culture is applied to the oil-polluted area in the form of a biological preparation comprising the above-cited strain in admixture with a mineral fertilizer containing nitrogen in the nitrate form. The amount of the culture to be applied is at least 1xc2x7104 cells/mg, where the flow rate of an aqueous mixture is from 0.5xc2x7to 1.0 liter per square meter.
However, the degree of recovery from various kinds of oil attained when using the above said bacterial strain also remains insufficient. Moreover, the application of this strain has certain limitations. This is associated with the fact that the strain grown on a standard carbohydrate substrate hardly adapts itself to new conditions, which means the necessity of using new substrates from soil. Besides, the said strain Pseudomonas putida-36 possesses hemolytic and gelatinizing activity which prevents the development of the process of natural self-recovery by means of microorganisms. Furthermore, the use of the nitrate forms of nitrogen when applying the bacterial culture into soil results in an additional contamination of the environment with toxic nitrate ions.
There has been known a pool of microorganisms Rhodococcus sp. Bkmacxe2x80x941500D, Rhodococcus maris VKM AC-1501D, Rhodococcus crythropolis VKM AC-1502D, Pseudomonas stutzeri VKM B- 1972D, Candida sp. VKM-Y-2778D and a biological preparation produced using this pool for cleaning water and soil polluted with crude oil and oil products.
However, the known pool and biological preparation are insufficiently effective when cleaning oil-polluted media and, moreover, characterized by a complicated production method because of a great number of various microorganisms constituting the pool. The industrial employment of this biological preparation comprising the said pool is difficult for cleaning purposes, since five strains constituting the pool differ very much in their physiological features and have different growth characteristics.
A known process for producing a biological preparation based on this pool comprises preparation of a solution of a liquid nutrient medium containing the sources of nitrogen, phosphorus, trace elements and liquid paraffins as a carbon source, followed by cultivation, for example in a fermenter, of an inoculation medium in the form of the above-mentioned pool of microorganisms, at a temperature of 28xc2x0 C. and pH maintained at 6.5-7.0. In so doing, the process of cultivation is carried out under aerobic conditions, for example when the medium is bubbled with the air flow. Thereafter, a biomass to be cultured is concentrated by the separation method and dehydrated by means of the lyophilic or thermal-vacuum drying to a 10% humidity. A biological preparation thus obtained contains 4xc2x71011-4xc2x71012 living cells per 1 gram and comprises both lipophilic and halophilic microorganisms. This biological preparation is capable of oxidizing petroleum products both in the zone of contacting with water and directly in an oil film and capable of removing oil pollution not only in the fresh but also in the saline ecosystem.
A known method of using the biological preparation obtained in accordance with the above described process consists in the utilization of an aqueous suspension prepared on its basis, in order to spray said suspension onto water and soil medium or the surface of equipment polluted with oil and/or oil-products. In so doing, a base suspension of the biological preparation is initially produced, said suspension being in the form of a concentrated aqueous solution of the biological preparation with a nitrogen-phosphate additive. To prepare this suspension, water having a temperature of from +15xc2x0 C. to +30xc2x0 C. is used. A composition thus prepared is maintained for 12 hours under stirring and aerating, after which time a base suspension is ready. To prepare a working suspension, the base suspension is supplemented with further nitrogen-phosphate additive followed by its dilution with water to obtain a desired concentration depending on the type of a medium to be cleaned and a degree of its pollution with oil and/or oil-products (see, Application PCT/RU 94/00103, IPC C 02F 3/34, 1995).
It is, therefore, an object of the present invention to provide a new pool of microorganisms and a biological preparation based thereof, which would possess improved properties: concurrent with the active work on the biodegradation of oil, in particular directly in an oil film, and of oil-processing products, would posses a shorter development cycle of microorganism components and would provide the biodegradation of a broad range of hydrocarbons constituting oil-processing products, under elevated temperatures and within a wide range of salinity of a medium to be recovered, and to provide most efficient methods for producing and utilizing this biological preparation.
A biological preparation in accordance with the present invention is a biodegrading agent for oil pollution. It includes a new pool comprising the yeast strains Candida maltosa VKPM Y-2256 and VKPM Y-2257. The strains Candida maltosa VKPM Y-2256 and VKPM Y-2257 have been selected from analogs isolated from oil-impregnated soil.
The two C. maltosa yeast strains VKPM Y-2256 and VKPM Y-2257 were deposited with the All Russia Collection of Industrially Deployed Microorganisms (State Scientific Research Institute of Genetics, 1st Dorozhniy Proyezd, 1, GNII Genetikaxe2x80x94VKPM, 113545 Moscow, Russia) on Mar. 3, 1997 under the registration numbers Y-2256 and Y-2257. They possess the following morphological-and-culture and physiological-and-biochemical properties.
On wort agar: the cells are oval and short-oval. Size of cells is 3.0-7.5xc3x972.5-6.3 xcexcm; fission is fast. In two-day culture in malt wort it forms islands of light dull film, a white dense sediment. Malt agar: large colonies, up to 0.7 cm in diameter, white color, shiny with yellowish shade in the middle part of the colony. A colony surface is smooth with an outlined pattern; a colony edge is fringed or even.
In an oxygen-free habitat, it actively consumes glucose, Saccharose maltose, galactose, xylose; slightly worsexe2x80x94arabinose; does not assimilate lactose. Well assimilates the ammonium form of nitrogen; does not assimilate potassium nitrate. Grows at +5-+41xc2x0 C. and pH 3.0-9.0. The growth optimum is at +30-+34xc2x0 C. and pH 3.8-5.5.
The Strain Candida maltosa VKPM Y-2257
The cells are oval and round-oval; single, as well as in the form of small chains (from three or four cells). Size of cells is 3.0-7.8xc3x975.2-3.0 xcexcm. Does not form ascospores. Mediumxe2x80x94malt agar: colonies are round, smooth, cream-colored with a smooth surface and even edge. A colony center is elevated and slightly runs against the edge. In malt wort it forms a film; does not form rings. Sediment is dense, light.
Aerobic properties. Well ferments glucose, saccharose, galactose, trehalose; slowlyxe2x80x94maltose; does not ferment lactose and raffinose. Assimilates glucose, saccharose, maltose, galactose, xylose, trehalose, glycerol, adonitol, sorbitol, mannitol. Does not assimilate lactose, raffinose, ribose, erythritol, inositol, soluble starch. Well fixes the ammonium form of nitrogen. Does not fix potassium nitrate. Grows at +5--+41xc2x0 C. and pH 3.0-9.0. The growth optimum is at +30-+41xc2x0 C. and pH 3.5-5.5. The strains are stored at +18-+20xc2x0 C. on wort agar.